Thus, this invention relates generally to the modification of a plant phenotype by the regulation of plant gene expression. More specifically it relates to the control of fruit ripening by control of one or more than one gene which is known to be implicated in that process.
Two principal methods for the control of expression are known. These are referred to in take art as "antisense downregulation" and "sensedownregulation" or "cosuppression". Both of these methods lead to an inhibition of expression of the target gene.
Overexpression is achieved by insertion of one or more than one extra copies of the selected gene. Other lesser used methods involve modification of the genetic control elements, the promoter and control sequences, to achieve greater or lesser expression of an inserted gene.
In antisense downregulation, a DNA which is complementary to all or part of the target gene is inserted into the genome in reverse orientation and without its translation initiation signal. The simplest theory is that such an antisense gene, which is transcribable but not translatable, produces mRNA which is complementary in sequence to mRNA product transcribed from the endogenous gene: that antisense mRNA then binds with the naturally produced "sense" mRNA to form a duplex which inhibits translation of the natural mRNA to protein. It is not necessary that the inserted antisense gene be equal in length to the endogenous gene sequence: a fragment is sufficient. The size of the fragment does not appear to be particularly important. Fragments as small as 40 or so nucleotides have been reported to obtain the inhibitory effect. However, it has to be said that fewer nucleotides may very well work: a greater number, up to the equivalent of full length, will certainly work. It is usual simply to use a fragment length for which there is a convenient restriction enzyme cleavage site somewhere downstream of fifty nucleotides. The fact that only a fragment of the gene is required means that not all of the gene need be sequenced. It also means that commonly a cDNA will suffice, obviating the need to isolate the full genomic sequence.
The antisense fragment does not have to be precisely the same as the endogenous complementary strand of the target gene. There simply has to be sufficient sequence similarity to achieve inhibition of the target gene. This is an important feature of antisense technology as it permits the use of a sequence which has been derived from one plant species to be effective in another and obviates the need to construct antisense vectors for each individual species of interest. Although sequences isolated from one species may be effective in another, it is not infrequent to find exceptions where the degree of sequence similarity between one species and the other is insufficient for the effect to be obtained. In such cases, it may be necessary to isolate the species-specific homologue. Antisense downregulation technology is well-established in the art. It is the subject of several textbooks and many hundreds of journal publications. The principal patent reference is European Patent No. 2540,208 in the name of Calgene Inc. There is no reason to doubt the operability of antisense technology. It is well-established, used routinely in laboratories around the world and products in which it is used are on the market. Both overexpression and downregulation are achieved by "sense" technology. If a full length copy of the target gene is inserted into the genome then a range of phenotypes is obtained, some overexpressing the target gene, some underexpressing. A population of plants produced by this method may then be screen and individual phenotypes isolated. As the antisense, the inserted sequence is lacking in a translation initiation signal. Another similarity with antisense is that the inserted sequence need not be a full length copy. Indeed, it has been found that the distribution of over- and under-expressing phenotypes is skewed in favour of underexpression and this is advantageous when gene inhibition is the desired effect. For overexpression, it is preferable that the inserted copy gene retain its translation initiation codon. The principal patent reference on cosuppression is European Patent No. 465,572 in the name of DNA Plant Technology Inc. There is no reason to doubt the operability of sense/cosuppression technology. It is well-established, used routinely in laboratories around the world and products in which it is used are on the market.
Sense and antisense gene regulation is reviewed by Bird and Ray in Biotechnology and Genetic Engineering Reviews 9:207-227 (1991). The use of these techniques to control selected genes in tomato has been described by Gray et al., Plant Molecular Biology, 19 69-87 (1992).
Gene control by any of the methods described requires insertion of the sense or antisense sequence, with appropriate promoters and termination sequences containing polyadenylation signals, into the genome of the target plant species by transformation, followed by regeneration of the transformants into whole plants. It is probably fair to say that transformation methods exist for most plant species or can be obtained by adaptation of available methods.
For dicotyledonous plants the most widely used method is Agrobacterium-mediated transformation. This is the best known, most widely studied and, therefore, best understood of all transformation methods. The rhizobacterium Agrobacterium tumefaciens, or the related Agrobacterium rhizogenes, contain plasmids which, in nature, cause the formation of disease symptoms, crown gall of hair root tumours, in plants which are infected by the bacterium. Part of the mechanism employed by Agrobacterium in pathogenesis is that a section of plasmid DNA which is bounded by right and left border regions is transferred stably into the genome of the infected plant. Therefore, if foreign DNA is inserted into the so-called "transfer" region (T-region)in substitution for the genes normally present therein, that foreign gene will be transferred into the plant genome. There are many hundreds of references in the journal literature, in text books and in patents and the methodology is well-established.
The effectiveness of Agrobacterium is restricted to the host range of the microorganism and is thus restricted more or less to dicotyledonous plant species. In general monocotyldonous species, which include the important cereal crops, are not amenable to transformation by the Agrobacterium method. Various methods for the direct insertion of DNA into the nucleus of monocot cells are known.
In the ballistic method, microparticles of dense material, usually gold or tungsten, are fired at high velocity at the target cells where they penetrate the cells, opening an aperture in the cell wall through which DNA may enter. The DNA may be coated on to the microparticles or may be added to the culture medium.
In microinjection, the DNA is inserted by injection into individual cells via an ultrafine hollow needle.
Another method, applicable to both monocots and dicots, involves creating a suspension of the target cells in a liquid, adding microscope needle-like material, such as silicon carbide or silicon nitride "whiskers" and agitating so that the cells and whiskers collide and DNA present in the liquid enters the cell.
In summary, the, the requirements for both sense and antisense technology are known and the methods by which the required sequences may be introduced are known. What remains, then is to identify genes whose regulation will be expected to have a desired effect, isolate them or isolate a fragment of sufficiently effective length, construct a chimeric gene in which the effective fragment is inserted between promoter and termination signals, and insert the construct into cells of the target plant species by transformation. Whole plants may then be regenerated from the transformed cells.
Fruit ripening is a complex developmental process which has been extensively used as a model system to dissect genetically programmed organ differentiation. Studies with both non climacteric and climacteric fruits such as apples, bananas, tomatoes, pears, avocados and mangos, have provided evidence for differential gene expression during ripening. Several enzymes showing altered activities during ripening have been reported and the respective genes have been cloned. The function of many ripening related genes is still unknown.
Muskmelon (Cucumis mel L.) is an economically important fruit that has an associated climacteric rise in ethylene production during ripening. Studies in melon, as with other climateric fruits, have shown that ripening is related to an increase in ethylene synthesis. A cDNA clone from melon with homology to the ACC-oxidase (Aco1) from tomato, which catalyst the terminal step in ethylene biosynthesis, has been isolated and shown to increase during ripening. The most notable physiological changes in fruit ripening are the softening of the mesocarp tissue, the accumulation of pigments, the development of the characteristic aroma and the sweet taste. Softening of the mesocarp is related to modification of pectin and hemicellulosic polysacharides. In melon, these changes are believed to be caused mainly by .beta.-galactosidases, whereas polygalacturonase is important in tomato, avocado and pears. Other enzymes are also involved in cell-wall catabolism such as cellulase and xylanase. The change of colour in ripe fruits is usually due to carotenoid or anthocyanin accumulation and chlorophyll degradation. This has been studied in detail in tomato, which like melon, synthesises carotenoids during ripening. A cDNA clone with homology to tomato phytoene synthase, a key enzyme in the carotenoid pathway, has been isolated from melon and shown to be preferentially expressed during ripening. Sweetness is a characteristic attribute of ripe muskmelon and it is also used in quality evaluation. Sugar level appears to be regulated by the balance of invertases and synthases present in the fruit tissue. Ripe fruit aroma is associated with a mixture of over fifty compounds, some of which include thioesters. Production and release of aromatic volatiles are not well-understood. All these properties of the ripe fruit make it attractive to the consumer and their possible manipulation is scientifically and commercially interesting. There is, however, a need to identify additional genes involved in melon ripening.